Hitoshi Ohishi

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, which is coupled to the inhibitory cyclic AMP cascade, was investigated in the central nervous system of the adult rat by in situ hybridization. Transcripts of mGluR2 were specifically localized to neuronal cells of the brain. Although the hybridization signals were widely distributed in the brain, the most prominent expression of mGluR2 messenger RNA was seen in Golgi cells of the cerebellum. Marked expression of mGluR2 messenger RNA was further observed in the mitral cells of the accessory olfactory bulb, neurons in the external part of the anterior olfactory nucleus, and pyramidal neurons in the entorhinal and parasubicular cortical regions. The granule cells of the accessory olfactory bulb, and many pyramidal and non-pyramidal neurons in the neocortical, cingulate, retrosplenial and subicular cortices, were moderately labeled. All of the granule cells in the dentate gyrus were also labeled moderately, whereas no significant hybridization signals were detected in Ammons horn. In the basal forebrain regions, moderately labeled neurons were distributed in the triangular septal nucleus, in the lateral, basolateral and basomedial amygdaloid nuclei, and in the medial mammillary nucleus. Weakly labeled neurons were sparsely scattered in the striatum, globus pallidus, ventral pallidum and claustrum. The subthalamic nucleus was also labeled weakly. No significant labeling was found in the entopeduncular nucleus and substantia nigra. In the thalamus, moderately labeled neurons were distributed in the anterodorsal, anteromedial, ventromedial, intralaminar and midline nuclei; the ventrolateral part of the anteroventral nucleus and the rostral pole of the ventrolateral nucleus also contained moderately labeled neurons. No significant labeling was found in the thalamic reticular, submedius, ventroposterior, lateral geniculate and medial geniculate nuclei. In the lower brainstem, labeling was generally weak. No significant hybridization signals were found in the spinal cord. Some neurons in the inner part of the inner nuclear layer of the retina and some retinal ganglion cells were labeled moderately. The pattern of distribution of mGluR2 messenger RNA revealed in the present study indicates specific roles of mGluR2 in the glutamatergic system in the brain.

Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain

A trpE-fusion protein containing a C-terminal sequence of a rat metabotropic glutamate receptor, mGluR5, was used to produce an antibody. On immunoblot, the antibody specifically reacted with mGluR5 expressed in mammalian cells and rat brain. Immunohistochemical analysis revealed intense mGluR5-like immunoreactivity (LI) in the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, cerebral cortex, hippocampus, lateral septum, striatum, nucleus accumbens, inferior colliculus, and spinal trigeminal nuclei. The distribution pattern of mGluR5-LI corresponds very well with that of mGluR5 mRNA. Electron microscope analysis of the striatum revealed dense accumulation of immunoreaction products in dendrites which were often provided with asymmetrical synapses. These results suggest that mGluR5 is predominantly located in postsynaptic elements.

Differential plasma membrane distribution of metabotropic glutamate receptors mGluR1α, mGluR2 and mGluR5, relative to neurotransmitter release sites

Two group I metabotropic glutamate receptor subtypes, mGluR1 and mGluR5, have been reported to occur in highest concentration in an annulus surrounding the edge of the postsynaptic membrane specialisation. In order to determine whether such a distribution is uniform amongst postsynaptic mGluRs, their distribution was compared quantitatively by a pre-embedding silver-intensified immunogold technique at electron microscopic level in hippocampal pyramidal cells (mGluR5), cerebellar Purkinje cells (mGluR1 alpha) and Golgi cells (mGluR2). The results show that mGluR1 alpha, mGluR5 and mGluR2 each have a distinct distribution in relation to the glutamatergic synaptic junctions. On dendritic spines, mGluR1 alpha and mGluR5 showed the highest receptor density in a perisynaptic annulus (defined as within 60 nm of the edge of the synapse) followed by a decreasing extrasynaptic (60-900 nm) receptor level, but the gradient of decrease and the proportion of the perisynaptic pool (mGluR1 alpha, approximately 50%; vs mGluR5, approximately 25%) were different for the two receptors. The distributions of mGluR1 alpha and mGluR5 also differed significantly from simulated random distributions. In contrast, mGluR2 was not closely associated with glutamatergic synapses in the dendritic plasma membrane of cerebellar Golgi cells and its distribution relative to synapses is not different from simulated random distribution in the membrane. The somatic membrane, the axon and the synaptic boutons of the GABAergic Golgi cells also contained immunoreactive mGluR2 that is not associated with synaptic specialisations. In the hippocampal CA1 area the distribution of immunoparticles for mGluR5 on individual spines was established using serial sections. The results indicate that dendritic spines of pyramidal cells are heterogeneous with respect to the ratio of perisynaptic to extrasynaptic mGluR5 pools and about half of the immunopositive spines lack the perisynaptic pool. The quantitative comparison of receptor distributions demonstrates that mGluR1 alpha and mGluR5, but not mGluR2, are highly compartmentalised in different plasma membrane domains. The unique distribution of each mGluR subtype may reflect requirements for different transduction and effector mechanisms between cell types and different domains of the same cell, and suggests that the precise placement of receptors is a crucial factor contributing to neuronal communication.

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber—CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber—CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber—CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: A light and electron microscopic study

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant‐specific antibodies raised against C‐terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7‐like immunoreactivities (LI). The distribution patterns of mGluR7‐LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a‐LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a‐LI in the rat but not in the mouse. For both mGluR7a‐ and mGluR7b‐LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a‐LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a‐ and mGluR7b‐LI are observed not only in presumed glutamatergic axon terminals, but also in non‐glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non‐glutamatergic axon terminals in various CNS regions. J. Comp. Neurol. 393:332–352, 1998.

In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs.

1 Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co‐localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2 IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3 IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4 IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small‐ and large‐sized neurones were labelled but no labelling was found in the glia. Expression of PPT A mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor‐positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor‐positive neurones co‐expressed the EP1, EP3 and EP4 receptor, respectively. 5 These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex

The distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was immunohistochemically examined in the rat cerebellar cortex at both light and electron microscope levels. An antibody was raised against a fusion protein containing a C-terminal portion of mGluR2. On immunoblot, the antibody reacted with both mGluR2 and mGluR3 in rat brain. mGluR2/3 immunoreactivity was expressed in cell bodies, dendrites, and axon terminals of Golgi cells, as well as in presumed glial processes. Golgi axon terminals with mGluR2/3 immunoreactivity were often encountered in the vicinity of glutamatergic mossy fiber terminals. The results suggest that transmitter glutamate may exert control influences upon Golgi cells not only through dendritic mGluR2/3, but also through axonal mGluR2/3.

Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody

The distribution of a metabotropic glutamate receptor mGluR2 in the central nervous system was immunohistochemically examined in the rat and mouse with a monoclonal antibody raised against an N-terminal sequence of rat mGluR2 (amino acid residues 87-134). Neuronal cell bodies with mGluR2-like immunoreactivity (mGluR2-LI) were clearly shown in the horizontal cells of Cajal in the cerebral cortex, neurons in the triangular septal nucleus and medial mammillary nucleus, Golgi cells and the unipolar brush cells in the cerebellar cortex, and Golgi-like and unipolar brush-like cells in the cochlear nucleus. Neuropil was intensely immunostained in the accessory olfactory bulb, bed nucleus of the accessory olfactory tract, neocortex, cingulate cortex, retrosplenial cortex, subicular and entorhinal cortices, stratum lacunosum-moleculare of CA1 and CA3, molecular layer of the dentate gyrus, periamygdaloid cortex, basolateral amygdaloid nucleus, bed nucleus of the anterior commissure, caudate-putamen, accumbens nucleus, thalamic reticular nucleus, anteroventral and paraventricular thalamic nuclei, granular layer of the cerebellar cortex, anterior and ventral tegmental nuclei, granular layer of the cochlear nucleus, and parvicellular part of the lateral reticular nucleus. Many axons in the white matter and fiber bundles were also immunostained. No glial cells with mGluR2-LI were found. No particular species differences were found in the distribution pattern of mGluR2-LI between the rat and mouse. The results indicate that mGluR2 is expressed not only in somato-dendritic domain, but also in axonal domain of excitatory and inhibitory neurons.

Pre- and postsynaptic localization of a metabotropic glutamate receptor, mGluR2, in the rat brain: an immunohistochemical study with a monoclonal antibody

A monoclonal antibody against a metabotropic glutamate receptor, mGluR2, was produced by using a glutathione S-transferase (GST) fusion protein containing an N-terminal sequence of rat mGluR2. Intense mGluR2-like immunoreactivity (mGluR2-LI) was seen mainly in neuropil of the cerebral cortical regions, hippocampus, olfactory bulb, some diencephalic nuclei, dorsal cochlear nucleus and cerebellar cortex. In the cerebellar cortex, mGluR2-LI was seen only in Golgi cells. In Ammons horn, mGluR2-LI was marked in the stratum lucidum of CA3 and the stratum lacunosum-moleculare of CA1-CA3, but not detected in the stratum pyramidale. The results indicate that mGluR2 is located not only presynaptically but also postsynaptically.

Localization of the neuromedin K receptor (NK3) in the central nervous system of the rat

The distribution of the neuromedin K receptor (NK3; NKR) in the central nervous system was investigated in the adult rat by using in situ hybridization and immunohistochemical techniques. The rabbit anti‐NKR antibody was raised against a bacterial fusion protein containing a C‐terminal portion of NKR and affinity purified with a Sepharose 4B column conjugated to the fusion protein. Immunoblot analysis was performed to test the reactivity and specificity of the antibody. Crude membrane was prepared from cDNA‐transfected Chinese hamster ovary (CHO) cells expressing each of the rat NKR, substance P receptor (NK1; SPR), and substance K receptor (NK2; SKR) and from the hypothalamus, cerebral cortex, and cerebellum. Immunoreactive bands were observed specifically in the NKR‐CHO cells, hypothalamus, and cerebral cortex but not in the SPR‐ or SKR‐CHO cells, nor in the cerebellum. Molecular weights of the immunoreactive bands ranged from 73 to 89 kDa and from 59 to 83 kDa in the NKR‐CHO cells and tissues, respectively. The distribution of NKR‐like immunoreactivity coincided with that of NKR mRNA. The expression of NKR was indicated on neuronal cell bodies and dendrites. NKR was found to be expressed intensely or moderately in neurons in the glomerular and granule cell layers of the main olfactory bulb; glomerular and mitral cell layers of the accessory olfactory bulb; layers IV and V of the cerebral neocortex; medial septal nucleus; nucleus of the diagonal band; bed nucleus of the stria terminalis; globus pallidus; ventral pallidum; paraventricular nucleus; supraoptic nucleus; zona incerta; dorsal, lateral, and posterior hypothalamic areas; amygdaloid nuclei; medial habenular nucleus; ventral tegmental area; midbrain periaqueductal gray; interpeduncular nuclei; substantia nigra pars compacta; linear, median, dorsal, and pontine raphe nuclei; posteromedial tegmental nucleus; sphenoid nucleus; nucleus of the solitary tract; intermediate and rostroventrolateral reticular nuclei; and lamina II of the caudal spinal trigeminal nucleus and spinal dorsal horn. These findings are discussed in relation to the physiological functions associated with neuromedin K.